Atopic diseases such as asthma, allergic rhinitis, food allergies, anaphylaxis and eczema result from a complex interplay between environmental factors and genetic factors (Vercelli, et al., Int. Arch. Allergy Immunol. 124:20-24 (2001) and Patino and Martinez, Allergy 56:279-286 (2001)). Infants at risk for asthma and other atopic diseases demonstrate increased expression of Immunoglobulin E (IgE) and increased numbers of peripheral eosinophils (Martinez, et al., N. Engl. J. Med. 332:133-138 (1995)), reflecting increased expression of cytokines such as interleukin-4 (IL-4) and 13 (IL-13), denoted TH2 (T Helper 2) cytokines, and relatively decreased expression of cytokines such as interferon g and interleukin-12 (IL-12), denoted TH1 (T Helper 1) cytokines (Wills-Karp, et al., Nat, Rev. Immunol. 1:69-75 (2001)).
Influenza virus is increasingly identified as a public health concern capable of overwhelming the existing health care system in the event of a pandemic strain. Pandemic strains are capable of causing mortality and morbidity in healthy immuno-competent adults. A paradigm for emergence of pandemic influenza is the 1918 pandemic that occurred due to emergence of a novel influenza strain of the H1N5 subtype with increased human mortality and morbidity (Kash, et al., J. Virol. 78(17):9499-9511 (2004)). Another paradigm is the current outbreak of avian influenza of the H5N1 subtype circulating in Asia that may directly infect humans, also with significantly increased mortality relative to more typical human adapted strains (Nguyen-Van and Hampson, Vaccine 21(16):1762-1768 (2003)). Influenza viral pathogenesis is magnified by the viral strategy of rapid mutation, facilitated by a viral genome of multiple segments that can re-assort independently between strains in co-infected cells.
The rapid emergence of different antigenic determinants on viral coat proteins requires yearly adjustments of vaccine targets and insures that there will be years in which conventional vaccines are inadequate. While molecular biology will provide more rapid strategies for vaccine production, an opportunity exists for the development of therapies for those individuals for whom vaccine protection is inadequate either by augmentation of host immune mechanisms or by anti-viral strategies that do not rely on vaccines or host immune response. Concerns regarding emergence of a pandemic strain underscore the lack of an effective therapy for influenza and the limitations of existing vaccine strategies. Vaccines may be of little use in the very young and elderly, or in patients with asthma or other chronic respiratory disease (Christy, et al., Arch. Dis. Child 89(8):734-735 (2004); Tan, et al., Am. J. Med. 115(4):272-277 (2003); and Bueving, et al., Am. J. Respir. Crit. Care Med. 169(4):488-493 (2003)). Unfortunately, these are exactly the patients most at risk of death from influenza infection. Pregnant women are also a subgroup at particular risk of influenza infection since there exists a correlation between infection in the first trimester and brain disorders in the exposed fetus including schizophrenia or other neurological abnormalities that may manifest many years after birth (Brown, et al., Arch. Gen. Psychiatry 61(8):774-780 (2004) and Gorman, Time 164(7):80 (2004)).
A current paradigm proposes that atopic diseases result from an imbalance in cytokine expression with increased expression of TH2 cytokines and relatively decreased expression of TH1 cytokines imprinted in infancy or early childhood (Patino and Martinez, Allergy 56:279-286 (2001)). In support of this paradigm, therapies directed at restoring cytokine balance in early childhood can ameliorate or even prevent atopic disease (Kalliomaki, et al., Lancet 357:1076-1079 (2001) and Murch, Lancet 357:1057-1059 (2001)). Potent antihistamines or other anti-inflammatory medications given to children at risk for asthma significantly delayed the incidence of subsequent asthma in children with IgE-mediated allergy, apparently by preventing histamine and proallergic cytokine release from degranulation of mast cells (Anoymous, Pediatr. Allergy Immunol. 9:116-124 (1998); Moller, et al., J. Allergy Clin. Immunol. 109:251-256 (2002); and de Longueville, Pediatr. Allergy Immunol. 11(Suppl. 13):41-44 (2000)).
The cellular receptors for IL-4 and IL-13 share a common subunit termed the IL-4 receptor α chain, but differ in subunit shared with the IL-4 receptor α chain (Keegan, et al., PNAS USA 92:7681-7685 (1995) and Gessner and Rollinghoff, Immunobiology 201:285-307 (2000)). Because of receptor sharing, IL-4 and IL-13 share some common effects on target cells including promotion of IgE synthesis and eosinophil survival, but also different effects upon other target cells. For example, IL-4 receptors but not IL-13 receptors are readily detected on the surface of T lymphocytes although IL-13 receptors may nonetheless be expressed intra-cellularly (Graber, et al., Eur. J. Immuno. 28:4286-4298 (1998)). Conversely, IL-13 but not IL-4 expression seems to promote changes in epithelial tissue architecture and mucous expression in the lung (Kuperman, et al., Nat. Med. 8:885-889 (2002) and Wills-Karp, et al., Science 282:2258-2261 (1998). In humans, mutations in the shared IL-4 receptor α chain are associated with atopic disease, although not in all populations studied (Hackstein, et al., Immunogenetics 53:264-269 (2001); Hall, Respir. Res. 1:6-8 (2000); Hershey, et al., N. Engl. J. Med. 337:1720-1725 (1997); Howard, et al., Am. J. Hum. Genet. 70:230-236 (2002); Karp and Wills-Karp, Microbes Infect. 3:109-119 (2001); Mitsuyasu, et al., Nat. Genet. 19:119-120 (1998); Olavesen, et al., Immunogentics 51:1-7 (2000); and Risma, et al., J. Immunol. 169:1604-1610 (2002)). In murine models, knockout of the IL-4 receptor shared IL-4 receptor α chain and knockouts of the IL-4 receptor activated STAT-6 signaling factor almost completely eliminate the allergic phenotype although some atopic response can be rescued with prolonged allergic stimulation (Gessner and Rollinghoff, Immunobiology 201:285-307 (2000); Grunewald, et al., Int Arch Allergy Immunol 125:322-8 (2001); Nelms, et al., Annu Rev Immunol 17:701-38 (1999); Noben-Trauth, et al., Proc Natl Acad Sci USA 94:10838-43 (1997); Noben-Trauth, et al., Eur J Immunol 32:1428-33 (2002); Quelle, et al., Mol Cell Biol 15:3336-43 (1995); Shimoda, et al., Nature 380:630-3 (1996); So, et al., FEBS Lett 518:53-9 (2002); and Zhu, et al., J Immunol 166:7276-81 (2001)). Selective blockade of the IL-4/IL-13 receptor with a mutated IL-4 competitive peptide antagonist also blocked allergic sensitization in the mouse (Tomkinson, et al., J. Immunol. 166:5792-5800 (2001)).
These observations illustrate the importance of the IL-4/IL-13 signaling pathway as a target for pharmacologic intervention to prevent or treat allergic diseases. Knockout of the shared IL-4 receptor α chain required for both IL-4 and IL-13 eliminated both IgE production and asthma-like lung pathology, suggesting a unique role for IL-13 in asthma and some atopic skin diseases (Wills-Karp, et al., Science 282:2258-2261 (1998); Wills-Karp, Respir. Res. 1:19-23 (2000); and Herrick, et al., Clin. Invest. 105:765-775 (2000)). A recent clinical trial of a soluble fragment of the human shared IL-4 receptor α chain capable of binding IL-4 (but not IL-13) showed some effectiveness in severe asthmatics (Steinke and Borish, Respir. Res. 2:66-70 (2002)). Importantly, no adverse effects related to loss of IL-4 function were noted in the lung or systemically in these human subjects.
IL-4 and IL-13 are also required for systemic immunity to some bacterial and parasitic infections (Karp and Wills-Karp, Microbes Infect. 3:109-119 (2001); Mountford, et al., Infect. Immun. 69:228-236 (2001); and Mohrs, et al., J. Immunol. 162:7302-7308 (1999)), and receptor inactivation could result in increased infections in targeted tissues. Targeted inactivation of the IL-4 receptor α chain to particular tissues such as lung or other tissues such as the digestive tract where polymorphisms of the IL-4 receptor are associated with inflammatory bowel disease (Klein, et al., Genes Immun. 2:287-289 (2001)) could be of benefit to prevent systemic immunodeficiency. Systemic immuno-modulation via targeted inactivation of the IL-4 receptor α chain might also be of benefit under some circumstances since loss of the IL-4/IL-13 receptor prevents the onset of systemic autoimmune diabetes in the mouse (Grossman and Paul, Curr. Opin. Immunol. 13:687-698 (2001) and Radu, et al., PNAS USA 97:12700-12704 (2000) and some tumors are also responsive to IL-4 Strome, et al., Clin. Cancer Res. 8:281-286 (2002); Essner, et al., J. Gastrointest. Surg. 5:81-90 (2001); and terabe, et al., Nat. Immunol. 1:515-520 (2000)). IL4 has also been shown to differentially modulate HIV1 replication in primary cells of the monocyte/macrophage lineage. The imbalance of IL4/IL13 TH2 cytokines over TH1 cytokines is thought to facilitate replication of viruses including the HIV-1, Influenza, and Epstein-Barr virus.
It is therefore an object of the present invention to provide nuclease resistant EGS that provide a therapy for respiratory diseases, IL-4 and/or IL-13 dependent systemic diseases, and viruses.
It is further an object of the present invention to provide EGS that target proteins required for generation and modification of the immunoglobulin and T-cell repertoire.
It is further an object of the present invention to provide EGS that target viral replication.
It is further an object of the present invention to provide formulations for inhalation containing EGS and methods for treating inflammatory and related diseases utilizing such formulations.
It is further an object of the present invention to provide a formulation that inactivates IL-4 receptror α chain and methods of use thereof.